In recent years, various peptides have attracted attentions as a drug candidate or research tool. There have been various attempts to develop a peptide library and screen peptides having affinity with a target substance.
As a method of artificially constructing a peptide library, a method using chemical synthesis, a method using a biosynthetic enzyme of a secondary metabolite, a translation synthesis system, and the like have been used conventionally.
It is however difficult to enhance the diversity of a library in the method using chemical synthesis. In addition, it takes time for screening or analyzing the relationship between the structure and activity of a compound.
The method using a biosynthetic enzyme of a secondary metabolite, on the other hand, permits rapid and convenient construction or chemical conversion of an elaborate backbone that cannot be achieved by the chemical synthesis method. Since enzymes have substrate specificity, however, kinds of compounds that can be synthesized are limited. This method is therefore not suited for use in the construction of a large-scale compound library.
When a translation system is used, a peptide library rich in diversity can be constructed in a short time by constructing an mRNA library and translating it in one pot. By using this system in combination with an mRNA display method or the like, a nucleic acid molecule which is a genotype and a peptide which is a phenotype can be associated with each other. A peptide that binds to a desired target molecule can be speedily and conveniently searched from the library and concentrated. Although synthesis of a peptide library by using such a translation system has many advantages, it can produce only peptidic compounds.
In screening using a library, identification of a compound that inhibits a target substance having protease activity is often required. The library of peptidic compounds is however cleaved by protease so that compounds that inhibit the activity of a target substance cannot be screened efficiently.
Each peptide of the peptide library may be modified in vitro with a post-translational modification enzyme, but an enzyme having desired activity does not always have activity in vitro. Furthermore, the expressed peptide library must be purified before the reaction with an enzyme and in addition, substrate specificity of the enzyme must be investigated so that it is not easy to obtain a library composed of peptides having a desired structure.
When the presence or absence, or degree of modification of a library is not known, the library is regarded to be inferior in usefulness because it needs correlation analysis between structure and activity as in the chemical synthesis system.
Patellamide produced by Prochloron didemni, that is, endozoic algae of sea squirt is a low molecular cyclic peptide which is presumed to have various physiological activities. It is biosynthesized via a unique pathway with products of a pat gene cluster consisting of patA to patG. The pat gene cluster and biosynthesis pathway of it are schematically shown in FIG. 6.
In this biosynthesis, PatE peptide which is a patE gene product becomes a precursor. Since the patE gene has a hypervariable region (cassette region), the product of it constructs a natural combinatorial library.
The PatE peptide has, on both sides of the cassette region thereof, a recognition sequence by a post-translational modification enzyme. The proteins which serve as the post-translational modification enzyme are PatA, PatD, and PatG. PatD introduces an azoline backbone into Cys, Ser, and Thr in the cassette of PatE and converts Cys into a thiazoline backbone and Ser and Thr into an oxazoline backbone.
PatA cleaves the N-terminal recognition sequence of the cassette region of the PatE.
PatG is composed of two domains. An N-terminal oxidase domain converts an azoline backbone introduced by PatD into an azole backbone, that is, converts a thiazoline backbone into a thiazole backbone. A C-terminal peptidase domain macrocyclizes, while cleaving a C-terminal recognition sequence of the cassette region of PatE.
The cassette regions of the above-described natural PatE have following similarities: (i) they are composed of 7 or 8 residues, (ii) they tend to have Ser/Thr/Cys to be modified at the 2nd, 4th, 6th, or 8th positions from the N-terminal of the cassette region, (iii) the residues (Ser/Thr/Cys) to be modified are not adjacent to each other in most cases, and (iv) many of the residues other than Ser/Thr/Cys are hydrophobic residues such as Val, Ala, Ile, Phe, and Leu (M. S. Donia et al.; Non-patent Document 1).
These similarities were presumed to be necessary for it becoming a substrate of PatD or PatG, a post-translational modification enzyme. It is however not known which residue of Ser, Thr, and Cys has been modified or not modified and substrate specificity of PatD and PatG has not been elucidated yet.
The present inventors have found that some of azoline backbone introducing enzymes have azoline backbone forming activity also in vitro; the sequence of the cassette region which becomes a substrate of such an azoline backbone-introducing enzyme is not limited to that described in Non-patent Document 1 but the cassette region can have various sequences; an azoline compound library can therefore be constructed efficiently in one pot by expressing a PatE library in a cell-free translation system and then modifying it with the azoline backbone introducing enzyme; and such a library can be used also for screening using a target substance having protease activity. A schematic view of an azoline backbone formation reaction of such a substrate having a leader sequence is shown in FIG. 1A.
The present inventors have confirmed further that even when PatE has, instead of the leader sequence or recognition sequence thereof, a predetermined sequence different from the natural sequence, it may become a substrate of an azoline backbone introducing enzyme; and as shown in FIG. 1B, even when a peptide separate from a cassette-region-containing peptide is used as a leader sequence portion, presence of such peptide in a reaction system containing an azoline backbone introducing enzyme permits introduction of an azoline backbone into the cassette region (according to Patent Document 1).